Ebola viruses are among the most lethal human pathogens with mortality rates approaching 90% for the Zaire subtype. They are also a potential bioterrorism agent. Ebola virus infection causes a severe hemorrhagic disease in humans for which there are no therapeutic treatments nor protective vaccines currently available. For these reasons, Ebola virus is classified as a category A priority pathogen by NIH. Expressed on the virus and infected cell surface, the Ebola glycoproteins facilitate entry of the virus into host cells and also interact with the host cells in other important ways. The overall goal of this research program is to thoroughly characterize Ebola glycoprotein-host cell interactions to identify potential targets for therapeutic intervention and also to inform about the basic biology of Ebola replication. This research proposal focuses upon identification and analysis of host factors that interact in with Ebola GP and characterization of how Ebola glycoproteins affect the host cell. The surface glycoprotein (GP) of Ebola plays a key role in entry of the virus into the host and is believed to be responsible for attachment, receptor recognition, and membrane fusion. Definition of the host factors that are required for Ebola entry is essential for comprehending Ebola virus replication and pathogenesis. Endosomal cathepsins are the only currently known cellular factor required for Ebola entry. Specific aim 1 utilizes a series of genetic and biochemical experiments designed to identify important cellular Ebola entry factors. In addition to its function during viral entry, Ebola GP appears to interact with host cells in other important ways. Tetherin/BST-2 is a recently described intrinsic cellular antiviral factor that is active against a number of enveloped viruses and blocks release of mature viral particles. Our recent PNAS manuscript demonstrates that Ebola GP antagonizes the cellular anti-viral factor Tetherin to promote release of Ebola particles. Studies in Specific Aim 2 will examine the requirements for Ebola GP inhibition of this cellular anti-viral factor and will analyze the effects of GP upon Tetherin. In addition to roles in entry and virus release, Ebola GP expression in host cells causes apparent dramatic down modulation of surface proteins leading to loss of cellular adhesion and diminished surface levels of MHC and several other host proteins. Preliminary data suggests a model in which the mucin region in GP acts as a steric shield to block surface accessibility. Testing this model, defining the mechanism of GP-mediated surface protein down-regulation and analyzing the consequences of down- modulation on immune recognition comprise Specific Aim 3 of this proposal.